Clear cell renal carcinoma (CRCC) is the most common and aggressive form of kidney cancer, and is inherently resistant to therapy. CRCC is typically initiated by inactivation of the von Hippel Lindau (VHL) tumor suppressor gene, resulting in the constitutive activation of the hypoxia inducible factors, HIF-1 and HIF-2. CRCC lesions show evidence of a shift from high HIF-1 expression in early lesions, to high HIF-2 expression in advanced disease. The mechanism for this 'HIF switch' is unknown. However, high HIF-2 is associated with increased dysplasia and advanced disease, whereas HIF-1 is frequently lost in advanced CRCC. Hence the targeting of the HIF switch that leads to HIF-2 specific activation may be of therapeutic benefit. We have identified the hypoxia associated factor (HAF), as a mediator of the switch from HIF-1 to HIF-2 by selectively degrading HIF-1, and promoting HIF-2 transactivation. We show that HAF promotes HIF-2 specific activation in CRCC cells, and that high HAF expression predicts for significantly decreased progression-free survival in patients with metastatic CRCC. Hence, our hypothesis is that CRCC is initiated by pVHL loss-of-function, resulting in the 'HIF switch' from HIF-1 to HIF-2 specific transcription mediated by HAF, which drives CRCC progression. Hence, the targeting of the HAF/HIF-2 axis may be of therapeutic benefit for the treatment of CRCC. The overall goal of our studies is to identify the mechanisms driving the HIF switch, which may lead to new strategies for more effectively treating CRCC, and to determine whether specific inhibition of HIF-2 will yield increased therapeutic benefit. Hence, our first aim is to elucidate the molecular mechanisms regulating the switch to HIF-2, which include HAF SUMOylation and hydroxylation of unique poly-proline motifs within HIF-2. Our second aim is to investigate the contribution of the HAF/HIF-2 axis in promoting CRCC progression and resistance to therapy using in vitro and in vivo models, and clinical CRCC samples. Here, we will address the impact of HAF on the anti-tumor response of CRCC cells in vitro and in vivo, and on tumor growth/metastasis in orthotopic mouse models. We will also investigate the relationship between HAF and HIF levels to tumor stage and patient survival, in samples from patients with VHL disease, and within a multistage CRCC tumor microarray. Our third aim is to develop peptide-mimetic inhibitors of HAF/HIF-2 binding as a novel way to specifically inhibit HIF-2. Our lead 7 residue peptide specifically inhibits HIF-2 activity in CRCC cells. This peptide will form the basis for a small molecule peptide-mimetic, which we will use as a pharmacological probe to investigate the potential anti-tumor activity of HIF-2 inhibition.